Characterization of three human sec14p-like proteins: a-Tocopherol

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled α-tocopherol to mitochondria in the same order of magnitude as the human α-tocopherol transfer protein (α-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Genomics and virulence factors of Fusobacterium necrophorum

Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.